EP1712615A1 - In vitro Herstellung einer Zellpopulation mittels Nährzellen - Google Patents

In vitro Herstellung einer Zellpopulation mittels Nährzellen Download PDF

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Publication number
EP1712615A1
EP1712615A1 EP05290836A EP05290836A EP1712615A1 EP 1712615 A1 EP1712615 A1 EP 1712615A1 EP 05290836 A EP05290836 A EP 05290836A EP 05290836 A EP05290836 A EP 05290836A EP 1712615 A1 EP1712615 A1 EP 1712615A1
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EP
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Prior art keywords
cell population
cells
feeder cells
cell
nucleic acid
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EP05290836A
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English (en)
French (fr)
Inventor
Hervé GROUX
Françoise Cottrez
Hervé Bastian
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Institut National de la Sante et de la Recherche Medicale INSERM
Sangamo Therapeutics SA
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
TxCell SA
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Application filed by Institut National de la Sante et de la Recherche Medicale INSERM, TxCell SA filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Priority to EP05290836A priority Critical patent/EP1712615A1/de
Priority to PL06743329T priority patent/PL1869166T3/pl
Priority to US11/918,485 priority patent/US8722401B2/en
Priority to SI200631411T priority patent/SI1869166T1/sl
Priority to PCT/EP2006/061648 priority patent/WO2006108882A1/en
Priority to DK06743329.2T priority patent/DK1869166T3/da
Priority to CA002604697A priority patent/CA2604697A1/en
Priority to AU2006234338A priority patent/AU2006234338B2/en
Priority to JP2008510529A priority patent/JP5247431B2/ja
Priority to PT06743329T priority patent/PT1869166E/pt
Priority to ES06743329T priority patent/ES2389502T3/es
Priority to EP06743329A priority patent/EP1869166B1/de
Publication of EP1712615A1 publication Critical patent/EP1712615A1/de
Priority to CY20121100831T priority patent/CY1113321T1/el
Priority to JP2012278952A priority patent/JP2013116105A/ja
Withdrawn legal-status Critical Current

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C12N2500/95—Protein-free medium and culture conditions
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/53—CD2
    • C—CHEMISTRY; METALLURGY
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/50—Coculture with; Conditioned medium produced by invertebrate cells
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Definitions

  • the invention relates to a method for the in vitro production of a cell population P' from a cell population P, said production requiring the presence of at least one factor which is expressed by feeder cells, wherein a) feeder cells proliferate at a temperature T 1 , b) proliferated feeder cells are contacted with the cell population P, c) the cell mixture obtained at step (b) is cultivated at a temperature T 2 which is chosen such that the cell population P proliferates and the feeder cells do not proliferate, the at least one factor being expressed by the feeder cells, and d) the cell population P' so produced is recovered.
  • the production consists in an expansion, the feeder cells are insect feeder cells and the cell population P to be expanded is a T lymphocyte population, preferably a Tr1 lymphocyte population.
  • Cell therapy is a group of new techniques that rely in particular on replacing diseased or dysfunctional cells with healthy, functioning ones. Moreover, cell therapy finds applications in immunotherapy, involving lymphocytes. These new techniques are being applied to a wide range of human diseases, including many types of cancer, neurological diseases such as Parkinson's and Lou Gehrig's Disease, spinal cord injuries, and diabetes, auto-immune or inflammatory diseases.
  • Cells are the basic building blocks of the human body and hold many of the keys to how the body functions. Cells serve both a structural and a functional role in the body, performing an almost endless variety of actions to sustain the body's tissues and organs. There are hundreds, perhaps thousands, of different specialized cell types in the adult body. All of these cells perform very specific functions for the tissue or organ they compose. These mature cells have been differentiated, or dedicated, to performing their special tasks.
  • Bone marrow transplants are an example of cell therapy in which stem cells in a donor's marrow are used to replace the blood cells of the victims of leukemia and other cancers.
  • Cell therapy is also being used in experiments to graft new skin cells to treat serious bum victims, and to grow new corneas for the sight-impaired. In all of these uses, the goal is for the healthy cells to become integrated into the body and begin to function like the patient's own cells.
  • many studies are currently under process for priming and expanding T lymphocytes to use them as an immunotherapeutic treatment for cancer and infectious diseases, among others.
  • a cell population of interest is its identification from a biological sample, based on the determination of the presence of markers specific for the cell population in question, and then to proceed to its enrichment by eliminating cells that do not express the specific markers.
  • a method does not provide a sufficient quantity of cells for therapy or research purposes.
  • a cell production system wherein said cells may be differenciated and/or expanded, such as a cell expansion system capable to maintain exponential growth of a cell population for at least two or three months in vitro, and to have a very well characterized cell population for injection purposes, in contrast to a mixed cell population enriched with the required cells but contaminated with cells which may have adverse effects.
  • beads or plates coated with anti-CD3 and anti-CD28 antibodies cannot support long-term growth of purified CD8+ T cells, and include other limitations, such as the high cost of the beads, the labor intensive process involved in removing the beads from the culture medium before infusion, and the fact that the bead based system is restricted by a need for GM (Good Manufacturing) quality control approval before the start of each application.
  • GM Good Manufacturing
  • the american patent application published on August 7., 2003 with the number US 2003/0147869 discloses the use of aAPCs engineered by the inventors to mimic dendritic cells in their ability to stimulate rapid CTL growth.
  • the K562 erythromyeloid cell line is used because it (1) is of human origin; (2) lacks MHC class I and II molecules to avoid allogeneic response ; (3) grows well using serum free medium; (4) has been extensively used in the literature (over 5700 references); (5) has been characterized cytogenetically; and (6) has been approved for phase I clinical trials.
  • eucaryotic cells rather than procaryotic cells, are usually preferred since expression of eucaryotic proteins in eucaryotic cells can lead to partial or complete glycosylation and/or formation of relevant inter-or intra-chain disulfide bonds of a recombinant protein.
  • a major drawback correlated with the use of such aAPCs is that it is necessary to proceed to their irradiation before contacting them with the cell population to be expanded, in order to stop their growth. This irradiation requires to stimulate repeatedly the cell population to be expanded, and leads to the eventual introduction of irradiated aAPC into the clinical setting. Furthermore, the irradiation of aAPC can lead to genetic mutations, which can lead to the production of non-desirable factors. Such mutations may not be controlled and it is not possible to be totally sure that the proliferation has stopped the proliferation of all the aAPC. Another drawback correlated with the use of eucaryotic aAPC is that these cells may allow the proliferation of eukaryotic viruses present in the cell population to be expanded.
  • the present inventors have surprisingly discovered that it was possible to produce a cell population P' from a cell population P using a feeder cell system which is different from the current aAPC system.
  • a feeder cell system consists in feeder cells expressing factor(s) allowing production of the cell population P', wherein the culture temperature of the feeder cells (T 1 ) is different from that of the cell population P (T 2 ) from which the cell population P' is to be produced.
  • Feeder cells are firstly cultivated at a temperature T 1 in a culture medium Mf, they are then contacted with the cell population P contained in a culture medium Mp. When the feeder cells are contacted with the cell population P, they may be cleared or not of their culture medium Mf.
  • the culture medium Mp does not initially contain the at least one factor.
  • the obtained mixture of feeder cells, cell population P and culture medium Mp is then cultivated at a temperature T 2 .
  • the at least one factor is expressed by the feeder cells and is thus then contained in the culture medium Mp.
  • the cell population P proliferates, but not the feeder cells.
  • the cell population P' which is thus produced is finally recovered.
  • This new method thanks to the change of temperature, allows to avoid the irradiation of the feeder cells.
  • Such a feeder cell system allows the expansion and/or the differentiation of a cell population P in order to produce an expanded and/or differenciated cell population P'.
  • a method for the in vitro production of a cell population P' from a cell population P in a culture medium Mp, wherein said production requires the presence of at least one factor in said culture medium comprising the following steps:
  • the ratio [feeder cells : cell population P] is indifferent when adding the feeder cells to the cell population P (step (b)).
  • this ratio may be [1:1].
  • the cell population P may be of any living organism origin such as fishes, or preferably of mammal origin, such as humans, dogs, cats, mice, rats, and transgenic species thereof
  • mammals within the scope of the invention include animals of agricultural interest, such as livestock and fowl.
  • Feeder cells may be of any type, provided that they do not proliferate at the culture temperature of the cell population P (T 2 ).
  • the skilled person who is with wide experience of cell culture knows the specific conditions to be used, in particular the culture temperatures T 1 and T 2 of each of feeder cell population and cell population P from which the cell population P' is produced.
  • the culture media Mf and Mp may be of any kind, provided that they are appropriate for said feeder cell and said cell population types, and will be easily selected by the skilled person (Schneider's medium, ).
  • production encompasses the expansion and/or the differentiation of the cell population P.
  • the production of the cell population P' from the cell population P consists in an expansion.
  • the terms “expansion”, “proliferation” and “growth” may be employed in an interchangeable way and refer to the increasing number of cells in a cell population.
  • the expressions “expansion cell system”, “expansion feeder cell system” and “cell factory” refer indifferently to a device including feeder cells of the present invention.
  • the cell population P is expanded exponentially.
  • Methods for monitoring expansion of cell populations are well known by the skilled person such as, for example, microscopic inspection, by the use of an electronic particle counter, or indirectly by measuring the incorporation of radioactive precursors.
  • the most common assay for cell proliferation is the incorporation of 3H-thymidine into cellular DNA.
  • the change of the yellow, water soluble dye 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl) tetrazolium bromide (MTT) into a violet, insoluble product (MTT-formazan by the succinate dehydrogenase present in the cell mitochondria (Amersham Biosciences Corp., US, etc.), or the CFSE (carboxyfluorescein diacetate succinimidyl ester) method may also be used.
  • the step (b) of contacting the feeder cells with the cell population P, and the step (c) of cultivating the mixture at the temperature T 2 are usually simultaneous steps: before contacting, the feeder cells and the cell population P are cultivated separately, respectively one at the temperature T 1 in the culture medium Mf and the other at the temperature T 2 in the culture medium Mp. Then, the feeder cells "alone", or the culture medium Mf containing the feeder cells, is/are contacted with the cell population P which is present in its culture medium Mp and which is being cultivated at the temperature T 2 . Consequently, the feeder cells pass immediately from the temperature T 1 to the temperature T 2 and stop to proliferate, unlike the cell population P, from which the cell population P' is produced thanks to the at least one factor which is expressed by the feeder cells.
  • the feeder cells die during step (c) because of the temperature T 2 which is no more appropriate for feeder cell culture.
  • the cell membrane fragments of the feeder cells that result from death of said cells are eliminated at step (d).
  • the obtained culture medium Mp is composed of a mixture of the obtained cell population P', viable feeder cells and optionally cell membrane fragments of the feeder cells, and the cell population P' has to be recovered at step (d).
  • a recovery can be made by separating the cell population P' from the viable feeder cells and optionally said cell membrane fragments using any appropriate separation method well known by the man skilled in the art, such as for example flow cytometry using a specific labelled ligand capable to bind at the surface of the feeder cells or a cell surface protein of the cell population P'.
  • Other methods may also be employed, such as washing methods and/or centrifugation such as density gradient centrifugation using separation media like Ficoll®, such a centrifugation being an appropriate method for eliminating cell membrane fragments.
  • the at least one factor is selected from the group comprising factors anchored to the cell membrane of the feeder cells or factors secreted by said feeder cells. More advantageously, said at least one factor interacts with a cell surface protein of the cell population P. Of course, said at least one factor may also interact with a cell surface protein of the cell population P' which is obtained during step (c).
  • the feeder cells When the feeder cells are cultivated at step (a), they express said at least one factor either at their cell membrane surface or in the culture medium Mf.
  • the "membrane factor” is already anchored to the feeder cell membrane, but the “secreted factor” may be eliminated if the feeder cells are previously cleared of their culture medium Mf.
  • both of the "membrane factor” and the “secreted factor” are expressed by the feeder cells at step (c), even if the feeder cells no more proliferate, and until death of said feeder cells. It is even possible that the "membrane factor” anchored to the cell membrane fragments of the dead feeder cells still play a role in the production of the cell population P'.
  • the cell population P from which the cell population P' is produced has cell surface proteins which are implicated in the cell signals allowing production of said cell population P'.
  • Such cell surface proteins are activated thanks to specific ligands, or factors, which are provided in the present invention by the feeder cells:
  • protein protein
  • polypeptide peptide
  • peptide employed in the present application refer indifferently to a molecule formed by the union in a long chain of smaller elements, the amino acids.
  • a “protein complex” refers herein to the union of at least two long chains of amino-acids.
  • the feeder cells are recombinant cells and contain an heterologous nucleic acid encoding said at least one factor.
  • recombinant cell or " recombinant feeder cell” refer to the introduction in said cells of an heterologous nucleic acid encoding the at least one factor.
  • Such an introduction encompasses a variety of techniques useful for introduction of nucleic acids into feeder cells including electroporation, calcium-phosphate precipitation, DEAE-dextran treatment, lipofection, microinjection and infection with viral vectors.
  • electroporation calcium-phosphate precipitation
  • DEAE-dextran treatment DEAE-dextran treatment
  • lipofection lipofection
  • microinjection microinjection and infection with viral vectors.
  • suitable methods are very well known by the skilled person, and can be found for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989 )).
  • the nucleic acid to be introduced may be, for example, DNA encompassing the gene(s) encoding the factor(s) susceptible to interact with (a) cell surface protein(s) of the cell line to be produced, genomic DNA fragment, sense strand RNA or a recombinant expression vector containing a cDNA encoding such gene(s).
  • the heterologous nucleic acid can encode the full length factor or alternatively it can encode a peptidic fragment thereof that is sufficient to allow the production of the cell population in accordance with the present invention, when introduced into the feeder cells.
  • the nucleic acid can encode the natural ligand (co-stimulatory protein) of the cell surface protein of the cell line to be produced, or a fragment thereof, or a modified form of the ligand or fragment thereof.
  • the invention is intended to include the use of fragments, mutants, or variants (e.g., modified forms) of the factor that retain the ability to enhance the production of the cell line.
  • a "variant" of the factor means a protein that shares a significant homology with the natural ligand and is capable of effecting cell line production.
  • biologically active or biologically active form of the protein include forms of factors that are capable of effecting cell line production.
  • One skilled in the art can select such variants of factor based on their ability to enhance cell production upon introduction of a nucleic acid encoding the factor in the feeder cells.
  • variants of factor that have amino acid substitutions, deletions and/or additions as compared to the naturally occurring amino acid sequence of a comparable native factor, yet still retain the functional activity of the natural form of the factor as described herein are also encompassed by the invention.
  • Such variants may contain for example conservative amino acid substitutions in which amino acid residues are replaced with amino acid residues having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta.-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains
  • the nucleic acid is in a form suitable for expression of the factor(s) in which it contains all of the coding and regulatory sequences required for transcription and translation of a gene, which may include promoters, enhancers and polyadenylation signals, and optionally sequences necessary for transport of the factor to the surface of the feeder cells, including N-terminal signal sequences. Regulatory sequences can also be selected to provide constitutive or inducible transcription.
  • the expression of the factor at the surface of the feeder cell can be confirmed by immunofluorescent staining of the cells. For example, cells may be stained with a fluorescently labeled monoclonal antibody reactive against the co-stimulatory molecule or with a fluorescently labeled soluble receptor which binds the factor.
  • the skilled person who knows very well the factors to be expressed by the feeder cells, also knows appropriate monoclonal antibodies which recognize factors expressed by the feeder cells. Alternatively, labeled soluble ligand proteins which bind to the factors can be used to detect their expression on the feeder cell surface.
  • the techniques and devices employed for detecting immunofluorescent stained cells are very well known by the skilled person ; preferably, a fluorescence-activated cell sorter (FACS) is used for detection.
  • FACS fluorescence-activated cell sorter
  • nucleic acid encoding a factor When the nucleic acid encoding a factor is operably linked to regulatory elements it is typically carried in a vector, including for example plasmids and viruses.
  • a nucleic acid comprising a nucleotide sequence encoding a factor of the present invention operably linked to regulatory control elements is also referred to herein as an "expression vector".
  • Expression vectors will be chosen relative to the feeder cell type to be transformed.
  • the feeder cells are drosophila insect feeder cells
  • drosophila constitutive vectors available for expression of proteins in cultured insect cells include the pAc series ( Smith et al., (1983) Mol. Cell Biol. 3:2156-2165 ) and the pVL series ( Lucklow, V. A., and Summers, M. D., (1989) Virology 170:31-39 ).
  • the feeder cells are insect feeder cells.
  • any appropriate insect feeder cell may be used in the present invention, provided that it fulfills the above mentioned conditions. It may be for example insect feeder cells of the Sf9 (among others deposited at the ATCC with the number CRL 1711 or at the DSMZ with the number ACC 125, and marketed by BD Biosciences Pharmingen, US), Sf21 (among others deposited at the DSMZ with the number ACC 119, and also marketed by BD Biosciences Pharmingen, US) or the S2 cell line.
  • the insect feeder cells are from the S2 drosophila cell line.
  • the S2 drosophila cell line is well known by the man skilled in the art, and has been widely disclosed in the prior art.
  • the S2 drosophila cell line is commercially available (Invitrogen, France, etc%), and has been deposited in particular at the German collection of micro-organisms and culture cells DSMZ ("Deutsche Sammlung von Mikroorganismen und Zellkulturen") with the number ACC 130, and disclosed in Schneider, J Embryol Exp Morphol, 27:1972, 353 ; it has also been deposited at the American type culture collection ATCC with the number CRL 1963.
  • the insect feeder cells are from the S2 drosophila cell line deposited on March 25., 2005 at the National Collection of Micro-organisms Cultures (CNCM, Pasteur Institute, Paris) under the number I-3407.
  • the cell population P is a mammal cell population.
  • feeder cells which may be used when said cell population P is a mammal cell population may be insect feeder cells or plant feeder cells.
  • the feeder cells are insect feeder cells, T 1 is inferior to T 2 and T 2 is at least about 35°C.
  • At least about 35°C means that the temperature may vary from 0.1°C below 35°C (from 34.9°C to 35°C). The skilled person is anyway aware of such minimal variations of temperature.
  • a great advantage provided by the use of insect feeder cells when a mammal cell population P' is to be produced is that (1) feeder cells and mammal cells do not proliferate at the same temperature (T 1 is inferior to T 2 and T 2 is at least about 35°C), and (2) mammal viruses do not proliferate in insect feeder cells, thus avoiding the possible virus contamination of the mammal cell population P/P' from the feeder cells.
  • the culture medium Mp is a serum-free culture medium.
  • Media exempt from any biological contaminant such as commercially available serum-free culture media (XVIVO-15 from BioWhittaker, Walkersville, MD ; AIM V medium from Invitrogen, etc%), are preferred.
  • the culture medium Mf is a serum-free culture medium.
  • Media exempt from any biological contaminant such as for example well known and commercially available serum-free culture media (Schneider's medium without serum marketed by BioWhittaker, Walkersville, MD GIBCO® serum-free insect cell culture media such as SFM marketed by Invitrogen, or Insectagro® serum-free media marketed by Krackeler Scientific Inc., US, etc)., are preferred in order to avoid subsequent contamination of the cell population P.
  • the present invention encompasses cell populations P of any types, such as for example immune system cells, skin cells, hepatic cells, bone marrow cells, stem cells, islet cells, fibroblasts, etc...
  • immune system cells are the "T cells", which are art-recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • a T cell can be a T helper (Th) cell, for example a T helper 1 (Th1) or a T helper 2 (Th2) cell.
  • the T cell can be a CD4+T cell, CD8+T cell, CD4+CD8+T cell, CD4-CD8-T cell, or any other subset of T cells such as for example a regulatory T (Tr1) cell.
  • T cells can be obtained from a number of sources, including peripheral blood leukocytes, bone marrow, lymph node tissue, spleen tissue, and tumors.
  • peripheral blood leukocytes are obtained from an individual by leukopheresis.
  • To isolate T cells from peripheral blood leukocytes it may be necessary to lyse the red blood cells and separate peripheral blood leukocytes from monocytes by, for example, centrifugation through, for example a PERCOLLTM gradient.
  • Cytotoxic T lymphocytes may be used in an immunotherapeutic treatment for cancer and infectious diseases.
  • dendritic cells have shown to offer a great potential in the treatment of cancer.
  • Stem cell therapy is emerging as a potentially revolutionary new way to treat disease and injury, with wide-ranging medical benefits. It aims to repair damaged and diseased body-parts with healthy new cells provided by stem cell transplants. Bone-marrow transplants used to treat leukaemia patients are a current form of stem cell therapy.
  • the instant in vitro production method allows to obtain such cells in a sufficient quantity for research or cell therapy applications.
  • the mammal cell population P is selected from the group comprising a T cell population, a dendritic cell population, an undifferenciated stem cell population, a predifferenciated stem cell population, a differenciated stem cell population, a skin cell population and a pancreatic islet cell population.
  • the mammal cell population P is a T cell population.
  • the feeder cells do not have any intrinsic class I and/or II major histocompatibility complex (MHC) molecule at their surface. It means that these cells do not naturally express MHC molecules, unless they have been genetically transformed. The absence of these intrinsic class I and/or II MHC molecules at the surface of the feeder cells is crucial to avoid an allogeneic response between the feeder cells and the mammal T cell population P. As a result, the feeder cells of the present invention may be used to expand a cell population P from any donor in a short time period.
  • MHC major histocompatibility complex
  • the feeder cells are cleared of their culture medium Mf at step (b).
  • the feeder cells express at least two factors, preferably 3 to 10 factors.
  • the choice of the at least two factors depends on cell surface proteins of the cell population P with which the factors have to interact. The skilled person knows which factors have to be expressed by the feeder cells for production of a cell population P' from a cell population P.
  • TCR/CD3 complex TCR for T cell receptor and CD for cell differentiation antigen
  • An anti-CD3 monoclonal antibody can be used to activate a population of T cells via the TCR/CD3 complex, advantageously a modified anti-CD3 antibody, wherein the modification of the anti-CD3 antibody consists in the replacement of the intracytoplasmic domain with a transmembrane domain, such that said modified anti-CD3 antibody anchors to the cellular membrane of the feeder cells and interacts with the CD3/TCR protein complex of the T cells.
  • co-stimulatory molecules proteins on the surface of T cells
  • co-stimulatory molecules proteins on the surface of T cells
  • co-stimulators have been implicated in regulating the transition of a resting T cell to blast transformation, and subsequent proliferation and differentiation.
  • co-stimulatory molecules proteins on the surface of T cells
  • co-stimulators in addition to the primary activation signal provided through the TCR/CD3 complex, induction of T cell responses requires a second co-stimulatory signal.
  • co-stimulatory or accessory molecule, CD28 is believed to initiate or regulate a signal transduction pathway that is distinct from those stimulated by the TCR complex.
  • the factor interacting with the CD28 protein present at the surface of the T cells and which is expressed by the feeder cells may be an anti-CD28 monoclonal antibody or a fragment thereof capable of crosslinking the CD28 molecule; in such a case, modification of the anti-CD28 monoclonal antibody can be envisaged by adding a transmembrane domain in order that it anchors to the cell surface of the feeder cells.
  • the natural ligand for CD28 is employed instead of the anti-CD28 monoclonal antibody, that is to say for example a member of the B7 family of proteins, such as B7-1 (CD80) and B7-2 (CD86) proteins.
  • IL-2 interleukin-2
  • the feeder cells are recombinant feeder cells expressing recombinant factors which interact with the following cell surface proteins of the T cell population:
  • the factors comprise :
  • the transmembrane domain which replaces the intracytoplasmic domain of the anti-CD3 antibody heavy chain is the transmembrane domain of the platelet derived growth factor (PDGF).
  • PDGF platelet derived growth factor
  • Tr1 regulatory cells Tr1 cells that exert important regulatory functions in various immuno-inflammatory diseases such as Crohn's disease ( H. Groux et al. Nature 1997, 389, 737-742 ), skin inflammation ( Foussat et al. 2003 J. Immunol. 171, 5018-5026 ), atherosclerosis ( Mallat et al. Circulation 2003, 108, 1232-1237 ) or multiple sclerosis (Barrat et al. 2002, 195, 603-616).
  • Crohn's disease H. Groux et al. Nature 1997, 389, 737-742
  • skin inflammation Foussat et al. 2003 J. Immunol. 171, 5018-5026
  • atherosclerosis Mallat et al. Circulation 2003, 108, 1232-1237
  • multiple sclerosis Barrat et al. 2002, 195, 603-616.
  • the international patent publication WO 2005/000344 discloses a method for identification of Tr1 lymphocytes in a biological sample, based on the determination of the simultaneous presence of the molecular group CD4, CD18 and/or CD 11 a, CD49b and, where appropriate, by the demonstration of an over-expression of genes encoding the proteins CD4, PSGL-1, PECAM-1 and alphaV/beta3. It is now possible to identify such Tr1 cells thanks to the above mentioned markers.
  • Tr1 cells can be identified and/or purified by Elisa, flow cytometry, immunoaffinity chromatography with antibodies directed against said markers, for example with :
  • Enrichment of CD3+CD4+CD18brightCD49b+ cells from lymphocytes can be performed with magnetic beads in two steps:
  • ELISA tests may also be used to measure IL-4, IL-10, and IFN-alpha expression.
  • the T cell population is a Tr1 cell population.
  • the inventors have discovered that it was necessary to activate the CD2 protein and the IL-2 and IL-4 receptors present at the surface of the Tr1 cells, in addition to the stimulation of the CD3/TCR complex and the CD28 potein required for expansion of a T lymphocyte population, in order to expand the Tr1 regulatory lymphocyte population.
  • the factors interact with the cell surface proteins of the T cell population as described above (CD3/TCR complex, CD28 protein, and optionally IL-2 receptor), and with the following additional cell surface proteins of the Tr1 cell population from which the Tr1 cell line is to be expanded :
  • the factors comprise those as described above (modified anti-CD3 antibody, and CD80 or CD86 protein, preferably CD80 protein) and the following additional factors :
  • a CD4+ T lymphocyte population may be expanded by interaction of factors expressed by the feeder cells with the usual CD3/TCR complex, the CD28 protein and the IL-2 receptor present at the surface of the CD4+ T lymphocytes.
  • factors have been previously described (anti-CD3 antibody, CD80 or CD86 protein and IL-2).
  • a CD4+ Th1 lymphocyte population may be expanded by interaction of factors expressed by the feeder cells with the usual CD3/TCR complex, the CD28 protein and the IL-2 receptor, plus the interleukin-12 (IL-12) receptor or the interferon (IFN) receptor and the lymphocyte function-associated antigen-1 (LFA-1), all these molecules being present at the surface of the CD4+ Th1 lymphocytes.
  • the factors anti-CD3 antibody, CD80 or CD86 protein and IL-2 may be used (see infra), plus the factors IL-12, which interacts with the IL-12 receptor, or the IFN-gamma, which interacts with the IFN receptor and the intercellular adhesion molecule-1 (ICAM-1), which interacts with LFA-1.
  • a CD8+ T lymphocyte population may be expanded by interaction of factors expressed by the feeder cells with the usual CD3/TCR complex, the CD28 protein and the IL-2 receptor, plus the CD40L (CD40 ligand), all these molecules being present at the surface of the CD8+ T lymphocytes.
  • the factors anti-CD3 antibody, CD80 or CD86 protein and IL-2 may be used (see infra), plus the factors CD40, which interacts with the CD40L, or the anti-CD40L, which interacts with the CD40L.
  • a stem cell population may be expanded by interaction of the stem cell factor (SCF) and/or the fetal liver tyrosine kinase-3 ligand (Flt3L) which are expressed by the feeder cells, with respectively the c-kit and/or Flt3 receptor.
  • SCF stem cell factor
  • Flt3L fetal liver tyrosine kinase-3 ligand
  • a fibroblast population may be expanded by interaction of the epidermal growth factor (EGF) which is expressed by the feeder cells, with the EGF receptor.
  • EGF epidermal growth factor
  • a dendritic cell population may be expanded by interaction of the granulocyte-macrophage colony-stimulating factor (GM-CSF), the IL-4 or IL-13 and optionally the tumor necrosis factor (TNF), which are expressed by the feeder cells, with the corresponding interacting molecules present at the surface of the dendritic cells.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL-4 or IL-13 optionally the tumor necrosis factor (TNF)
  • TNF tumor necrosis factor
  • the T cell population is an antigen-specific T cell population.
  • antigen in the expression "antigen-specific T cell population” refers to an immunogenic peptide.
  • Immunogenic peptides are non-pathogenic peptides or proteins that can bind to MHCII molecule of an individual and that is recognized by the T cell receptors of said individual.
  • the antigen is a non-allergic food antigen (ovalbumin, etc%) or a non-pathogenic bacterial antigen.
  • T lymphocytes are contacted with an antigen in a form suitable to trigger a primary activation signal in the T lymphocyte, that is to say the antigen is presented to the T lymphocyte such that a signal is triggered in the T cell through the CD3/TCR complex.
  • the antigen can be presented to the T cell in a soluble form (antigen coupled to a soluble MHC molecule, ...) or by an antigen presenting cell in conduction with an MHC molecule.
  • An antigen presenting cell such as a B cell, macrophage, monocyte, dendritic cell, Langerhan cell, or other cell which can present antigen to a T cell, can be incubated with the T cell in the presence of the antigen (for example a soluble antigen) such that the antigen presenting cell presents the antigen to the T cell.
  • a cell expressing an antigen of interest can be incubated with the T cell.
  • a tumor cell expressing tumor-associated antigens can be incubated with a T cell together to induce a tumor-specific response.
  • a cell infected with a pathogen for example a virus, which presents antigens of the pathogen can be incubated with a T cell.
  • the antigen-specific T lymphocyte population can be expanded in accordance with the method of the invention. The same applies for any sub-type of T lymphocyte population, in particular for a Tr1 lymphocyte population.
  • the antigen-specific T cell population is an antigen-specific Tr1 cell population.
  • Factors which are expressed by the feeder cells may be of any origin. Preferably, they are of the same origin than that of the mammal cell population P to be expanded. More advantegously, the cells of said mammal cell population P are human cells. Most preferably, the at least one factor is of human origin.
  • the light chain of the modified anti-CD3 antibody is encoded by the heterologous nucleic acid of sequence SEQ ID N°1, or any nucleic acid having at least 70 % of identity with SED ID N°1
  • the heavy chain of the modified anti-CD3 antibody is encoded by the heterologous nucleic acid of sequence SEQ ID N°2, or any nucleic acid having at least 70 % of identity with SED ID N°2.
  • the CD80 protein is encoded by the heterologous nucleic acid of sequence SEQ ID N°3, or any nucleic acid having at least 70 % of identity with SED ID N°3.
  • the CD86 protein is encoded by the heterologous nucleic acid of sequence SEQ ID N°4, or any nucleic acid having at least 70 % of identity with SED ID N°4. More preferably, the IL-2 is encoded by the heterologous nucleic acid of sequence SEQ ID N°5, or any nucleic acid having at least 70 % of identity with SED ID N°5. Even more preferably, the CD58 protein is encoded by the heterologous nucleic acid of sequence SEQ ID N°6, or any nucleic acid having at least 70 % of identity with SED ID N°6.
  • the IL-4 is encoded by the heterologous nucleic acid of sequence SEQ ID N°7, or any nucleic acid having at least 70 % of identity with SED ID N°7.
  • the IL-13 is encoded by the heterologous nucleic acid of sequence SEQ ID N°8, or any nucleic acid having at least 70 % of identity with SED ID N°8.
  • nucleic acid molecule having at least 70 % of identity with SEQ ID No. X refers to any sequence which has at least 70, 75, 80, 85, 90, 95 or 99 % of identity with said sequence SEQ ID No. X.
  • percentage of identity between two nucleic acids in the present invention, it is meant a percentage of identical nucleotides between the two sequences to compare, obtained after the best alignment ; this percentage is purely statistical, and the differences between the two sequences are randomly distributed and all along their length.
  • the best alignment or optimal alignment is the alignment corresponding to the highest percentage of identity between the two sequences to compare, which is calculated such as herein after.
  • the sequence comparisons between two nucleic acids are usually performed by comparing these sequences after their optimal alignment, said comparison being performed for one segment or for one "comparison window", to identify and compare local regions of sequence similarity.
  • the optimal alignment of sequences for the comparison can be performed manually or by means of the algorithm of local homology of Smith and Waterman (1981) (Ad. App. Math. 2:482 ), by means of the algorithm of local homology of Neddleman and Wunsch (1970) (J. Mol. Biol. 48:443 ), by means of the similarity research method of Pearson and Lipman (1988) (Proc. Natl. Acad. Sci. USA 85:2444 ), by means of computer softwares using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI).
  • the percentage of identity between two nucleic acid sequences is determined by comparing these two aligned sequences in an optimal manner with a "comparison window" in which the region of the nucleic acid sequence to compare may comprise additions or deletions with regard the sequence of reference for an optimal alignment between these two sequences.
  • the percentage of identity is calculated by determining the number of positions for which the nucleotide is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the "comparison window” and by multiplying the result obtained by 100, to obtain the percentage of identity between these two sequences.
  • the expanded cell population P' is recovered when all the feeder cells are dead, which allows firstly to obtain a larger expanded cell population P', and secondly to recover rapidly and easily the expanded cell population P' by eliminating the cell membrane fragments of the feeder cells, for example by washing methods and/or density gradient centrifugation, as disclosed above.
  • the T cell line is recovered at step (d) after having cultivated the T cell population at step (c) during at least 12 hours, advantageously 24 hours.
  • the present invention also encompasses the particular embodiment wherein a unique culture medium is used, and the method is as follows:
  • Figure 1 Analysis of human protein expression on S2 cell line.
  • Figure 2 Cartoon of engineered CF interacting with a CD4 +Tr1 cell
  • S2 cells were transfected with a membrane bound anti-CD3 mAb to engage the TCR/CD3 complex, CD80 and CD58 to add some costimulatory signals through interaction with CD28 and CD2 molecules respectively, and IL-2 and IL-4 to enduce cell growth.
  • Figure 3 Proliferation of T cells induced by CF cell line.
  • Tr1 cells lines L1 and L2
  • Tr1 clones C1 and C2
  • Proliferation of polyclonal PBLs, CD4 +T cells Tr1 cells lines (L1 and L2) or Tr1 clones (C1 and C2) stimulated with the cell factory was measured by [3H]thymidine incorporation between days 3 and 4 culture.
  • T cells were stimulated with CF cells as indicated, in the absence of exogeneous cytokines.
  • the cells were pulsed with [3H]thymidine and incubated for an additional 18 h before harvesting.
  • Counts per minute values are shown as mean s.e.m. from triplicate cultures.
  • Figure 4 Long-term growth of primary polyclonal human Tr1 cells stimulated with cell factory.
  • Tr1 cells were stimulated with CD3/28 beads plus exogeneous IL-2 and IL-4, CF' cells expressing OKT3, CD80 and CD58 but not IL-2 and IL-4 in the presence of exogeneous IL-2 and IL-4, or with the complete cell factory system without any exogeneous addition. T cells were stimulated with CF cells on days 0, 10, and 20 of culture.
  • Figure 5 Purity of T cells after co-culture with CF cell line.
  • T cells and after stimulation with CF cell line was assessed by staining for CD3, CD4 expression during the first seven days of culture. Gating on cell size/debris was not used in this experiment so as to represent all cells in the culture. Viable cells are indicated by gating on propidium iodide to exclude dead cells. Results are representative of >10 different experiments, each with a different donor.
  • CF stimulator cells were assessed by staining for CD4 and OKT3H expression during the first seven days of culture. Gating on cell size/debris was not used in this experiment so as to represent all cells in the culture. Viable cells are indicated by gating on propidium iodide to exclude dead cells. Results are representative of >10 different experiments, each with a different donor.
  • FIG. 7 Schematic representation of the experimental protocol used.
  • Figure 8 Isolation of OVA-specific Tr1 clones.
  • PBL stained with CFSE were stimulated with OVA, and stained with CD4 CD49b and CD18.
  • CD4 + CD49b + CD18 bright cells were gated and CFSE cells were sorted. Sorted cells were cloned to generate clone 1 and 2, the bulk population was stimulated with OVA and stained with IL-10 and IFN- ⁇ revealing a Tr1 phenotype.
  • Figure 10 Cytokine profile of OVA-specific T clones 1 and 2 after expansion on the cell factory for 70 days.
  • Cytokine were measured in the supernatants of the clones stimulated with OVA and autologous irradiated monocytes. Antigen-specific suppression was also examined by a transwell assay. Autologous PBLs were stimulated with anti-CD3 mAb in the bottom well, no cells, control CD4 T cells and the two clones were added in the top bascket and stimulated with anti-CD3 and autologous irradiated monocytes for CD4 cells or OVA and irradiated autologous monocytes for the two Tr1 clones. The entire protocol is representative of ten experiments, each from different donors.
  • CD80 biotinylated mouse-anti-human CD80 (B7-1), clone L307.4 (BD
  • CD80 mouse-anti-human CD80-PE (phycoerythrine) or FITC (fluorescein isothiocyanate), clone L307.4 (BD Biosciences Pharmingen)
  • CD58 mouse-anti-human CD58-PE or PECy5 (phycoerythrin-cyanin 5) (LFA-3) Clone 1C3 (BD Biosciences Pharmingen)
  • Cloning and construction of cell factory Human CD80, IL-2, IL-4 and CD58 were cloned from peripheral blood T lymphocytes (PBLs) obtained from a healthy donor into the pAC vector (Invitrogen) using an insect actin promotor ( Chung and Keller, Mol Cell Biol. 1990 Dec;10(12):6172-80 ; Chung and Keller, Mol Cell Biol.
  • CD4 + T-lymphocyte preparation and S2 cell culture Fresh peripheral blood lymphocytes were obtained by Ficoll hypaque centrifugation, and CD4 +T cells were purified by negative selection using anti-CD8 antibody (Becton Dickinson). All cultures were maintained in X-vivo without serum addition (BioWhittaker, Walkersville, MD). Human IL-2 (Chiron Therapeutics, Emeryville, CA) was added at 20 IU/mL where indicated, hIL-4 was used at 1 ⁇ g/mL (for comparing the biological advantage obtained when feeder cells express the interleukins with the results obtained when recombinant intereukins are added in the culture medium). S2 cells were maintained in Schneider medium without serum (BioWhittaker, Walkersville, MD).
  • Tr1 cells have distinct co-stimulation requirements for long-term growth
  • the inventors designed a cell-based system that could be genetically manipulated to express different co-stimulatory molecules and cytokines in addition to CD3/CD28 classical stimuli. They chose S2 cells because they do not express human HLA proteins that would promote allogeneic responses, and they could not be contaminated by human viruses (Fig. 1). Also, the eventual introduction of irradiated feeder cells into the clinical setting can be avoided because these cells which grow at 27°C are easily killed at 37°C and are propagated in serum-free medium.
  • the inventors transfected and then cloned S2 cells expressing the human CD80, the human CD58 and the two chains of an anti-hCD3 mAb to permit the stimulation of human Tr1 cells (CF') (Fig. 1). Similarly, they generated the CF line (Figs. 1, 2) by transfecting CF' cells with human IL-4 and IL-2 cDNA. Cultures were initiated by adding CF cells to fresh human CD4 +T cells prepared by negative selection (see Experimental Protocol).
  • CF cell line efficiently activate human polyclonal CD4 +T cells and Tr1 cells.
  • the cell factory was tested for its ability to stimulate the initial activation and proliferation of primary CD4 +T cells as well as Tr1 cell lines or Tr1 cell clones.
  • the different purified T cells were stimulated with the cell factory at an 1/1 ratio.
  • the inventors found that the initial rate of growth of the T cells stimulated with the cell factory was equivalent, as judged by [3H]thymidine incorporation (Fig. 3) with a slight enhancement of Tr1 cells proliferative response over other CD4 + T cells.
  • the inventors confirmed this observation by labeling fresh T cells with carboxyfluorescein diacetate succinimidyl ester (CFSE) and tracking cell division during the first five days of culture (data not shown). They also found that the cell-based system was more efficient than CD3/28 beads for the induction of proliferation and cell division of CD4 +T cells (data not shown). No proliferation was seen in when the cell factory, or CD4 +T cells incubated separately (Fig. 3 and data not shown). Thus, the requirements for the initial rounds of CD4 +T-cell proliferation was even more satisfactory with the cell factory as compared to CD3/CD28 stimulation provided in the context of polystyrene beads.
  • Tr1 cells permit long-term expansion of human Tr1 cells.
  • the inventors determined whether the cell factory was sufficient to maintain long-term propagation of Tr1 cells (Fig. 4).
  • Tr1 cells were stimulated with CF that secrete hIL-2 and IL-4, with CF' that do not secrete cytokine but with addition of exogeneous cytokines and, CD3/28 beads with exogeneous cytokines.
  • CD3/28 bead-stimulated cells failed to proliferate after the second stimulation, in agreement with previous studies.
  • Tr1 cells stimulated with CF' in the context of IL-2 and IL-4 added exogeneously entered into a plateau phase of the growth curve within two weeks of culture, and no additional net growth of cells occurred after re-stimulation.
  • Tr1 cell cultures were stimulated with the cell factory, they remained in exponential growth even after a third stimulation. This augmentation of long-term proliferation was reproducible, as the average increase in the total number of T cells was 810-fold higher in cultures stimulated with the cell factory than in cultures stimulated with CD3/28 beads in six independent experiments.
  • Phenotypic analysis of cultures showed a progressive enrichment for CD3 +CD4 +T cells after stimulation with the cell factory (Fig. 5).
  • the S2 cells rapidly disappeared from the cell culture, as evidenced by an inability to detect the cells expressing the anti-CD3 mAb by flow cytometry after seven days (Fig. 6); this finding was confirmed in large-scale experiments and also by RT-PCR for drosophila genes (data not shown).
  • the mixed T-cell and cell factory culture yields a population of pure T cells within one week.
  • the one antigen-specific Tr1 cell yielded 1.5 10 9 cells after one and an half month of culture, a number of cells sufficient for immunotherapy.
  • the substantial proliferative capacity of the Tr1 cells that remains after 30 days of culture suggests that these Tr1 could have substantial long-term engraftment potential after adoptive transfer.
  • the cell factory Compared with microspheric aAPCs, or other non cell based stimulation assay, the cell factory allows better formation of the immunological synapse as a result of the fluidity of the APC membrane. Furthermore, the present system employing S2 cells as the scaffold has several other advantages for use in the clinic: they lack MHC expression, are mycoplasma-free, do not require irradiation, do not allow expansion of mammalian viruses and have been adapted for growth in serum-free medium. In addition, this cell factory can be used "off the shelf' to expand populations of Tr1 cells from any donor.
  • the cell factory system is able to maintain exponential growth of Tr1 cells for at least two to three months in vitro. Based on a starting cell number of one antigen-specific Tr1 cells, the inventors obtained a sufficient number of Tr1 cells for therapy after only 30 to 45 days of culture. This efficacy allows for the first time the ability to use well characterized T cells clones for cell therapy. Therefore, only very well characterized cells will be injected in contrast to mixed cell population enriched with the required cells but contaminated with cells which will have at the least only no or adverse effects. Alternatively, this cell factory system could also be used with a MHC class II tetramer to enrich a population of antigen-specific population therefore accelerating the time to reach the number of 10 9 cells.
  • Tr1 cells retain a substantial replicative capacity after culture with the cell factory, even after reaching therapeutic numbers for clinical infusion.

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AU2011364392B2 (en) 2011-03-25 2017-03-02 Txcell Method for using regulatory T cells in therapy
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HUE060369T2 (hu) 2012-02-09 2023-02-28 Baylor College Medicine Peptidkeverékek széles spektrumú multivírus CTL-ek elõállítására
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US8722401B2 (en) 2014-05-13
CA2604697A1 (en) 2006-10-19
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WO2006108882A1 (en) 2006-10-19
JP5247431B2 (ja) 2013-07-24
US20090221070A1 (en) 2009-09-03
CY1113321T1 (el) 2016-04-13
JP2008535525A (ja) 2008-09-04
DK1869166T3 (da) 2012-09-24
EP1869166A1 (de) 2007-12-26
WO2006108882A8 (en) 2006-12-14
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